Project Summary: Enormous strides continue to be made in the design of nanoparticles as highly specialized therapeutics for achieving superior outcomes over standard pharmacological agents, the latter often associated with significant toxicity that limits treatment efficacy. While cancer immunotherapies have revolutionized the treatment of disease and shown therapeutic benefits in hard-to-treat cancers, these agents are limited, for example, by immune-related adverse events and off-target effects in immunosuppressive microenvironments. Novel, emerging anti-cancer strategies are therefore critically needed to overcome these limitations and improve durable response rates in combination with immune therapies. One promising strategy exploits the unique ?self- therapeutic? capabilities of the nanomaterials themselves ? the treatment of tumors without the need for cytotoxic drugs. These capabilities are governed by the intrinsic physico-chemical properties of these materials, which can lead to disruption of signal transduction pathways, cell cross-talk or invasion, and/or induced cell death programs within the tumor microenvironment (TME) ? providing unprecedented opportunities for combating disease. We have developed specialized ultrasmall fluorescent core-shell silica nanoparticles, Cornell prime dots (C' dots), with intrinsic therapeutic capabilities enabling a distinct combination of activities that (1) selectively and directly induce cancer cell death through the iron-dependent mechanism of ferroptosis and (2) modulate immune cells directly by priming T cells and polarizing macrophages toward a pro-inflammatory phenotype. As CD8+ T cells are known to also regulate ferroptosis during immunotherapy, such effects are expected to synergize with those induced by C' dots. A long-term goal of this proposal is to determine critical C' dot physico-chemical parameters responsible for maximizing responses to these intrinsic therapeutic activities. In Aim I, we will examine the extent to which changes in the structural properties of PEG-coated C' dots, plain or modified to specifically bind to melanocortin-1 receptor (MC1-R; a well-established target overexpressed by our syngeneic murine models and human melanomas), influence therapeutic efficacy in syngeneic melanoma models by modulating ferroptosis and the tumor microenvironment, in the presence and absence of checkpoint blockade. In Aim II, we will probe underlying mechanisms driving regulation of immune cell phenotype and/or induction of ferroptosis in vitro. The successful completion of the project will provide critical insights into (i) key structural parameters modulating the combined self-therapeutic activities of these particles related to their induction of ferroptosis and priming the tumor immune microenvironment; (ii) whether critical differences exist in particle characteristics needed to optimize these distinct activities; (iii) mechanisms underpinning these activities; and (iv) therapeutic strategies that maximize potent anti-tumor effects in syngeneic melanoma models by administering therapeutic doses of particles in tandem with checkpoint inhibitors (anti-PD-1 and anti-CTLA-4).